Molecular Identification Of The The Torreya Main Cultivars By Rapd

Torreya grandis is the special and precious economic tree with many uses in our country. Its whole body is just a gem and its seed is a well-known nut with a high economic value. In the recent years more and more improved breeds of Torreya grandis have been cultivated with the rapid productive development as one of trees with high economic benefit. But because of the lack of branches and nursery stocks of improved varieties, some cheats often happened from lawless persons by using the sham as the true. So at present the urgent problem during the course of producing and improving breeds is how to identify the ? improved breeds of Torreya grandis. Because the research on the breed classification of Torreya grandis is still limited to the research methods of morphology and anatomy, so it is very hard to find any article about the classification research at the level of DNA. In these ? experiments we try to perform the systematical analysis of assembling classes about the main cultivated breeds of Torreya grandis, and try to divide and identify the varieties and classes by using RAPD technology. The aim of this research is to provide data for the further deepen study of this precious tree species for the potential economic value. In this research the material for the analysis of RAPD is the endosperm DNA of Torreya grandis seed. there are eight breeds: qiefei, xuanwenfei, dayuanfei, xiaoyuanfei, xifei, mifei, zhimafei, chonggangfei. The DNA samples of endosperm of Torreya grandis seed which are extracted by using improved CTAB method are dissolved into TE buffer liquid for the purpose of determining its DNA density by using Du-640 nucleic acid and protein analytical instrument. The result indicates that the DNA samples content a high density DNA and considerable impurities such as protein, and its purity is not high. But the DNA samples can totally meet the need of RAPD analysis because RAPD doesn抰 need a high purity template DNA. For the purpose of improving the repeativeness of RAPD we probe for all procedures of RAPD analysis, especially for the reaction system and circular condition of RAPD during the course of the experiments. At last the optimal reaction conditions are established for the RAPD analysis of endosperm DNA of Torreya grandis. The reaction system of RAPD is: lOng/jil DNA template 2.0~il; lOx buffer l.Sixl; 2mM dNTPs 1.8j1l; 25mM MgC12 l.44iil; lOj.tM primer 1.26p1; 5U Tag polymerase 0.2pJ; ddH2O 6.8p1; total ISp]. the circular condition ofRAPD is: 9500, 3mm; one circle; 9400, 30s, 3600, 30s, 7200, 60s, 35 circles; 72 ~C 5mm. The DAN samples of eight man cultivated breeds of Torreya grandis are analyzed with RAPD, and 80 marker sites are found by using 23 primers that are screened from 520 random primers. The marker genotype of eight man cultivated breeds of Torreya grandis is formed and the molecular identification system is established on the base of it. Eight man cultivated breeds of Torreya grandis are molecularly classified into two classes after the marker genotype is transformed into data of 0-1 -2 kind which is analyzed with a assembling class software of 0-1-2 kind.